1. Field of the Invention
The present invention concerns lantibiotic mutants and chimeras of enhanced stability and activity, leader sequences for such lantibiotic mutants and chimeras, genes encoding such lantibiotic mutants and chimeras (both with and without the leader sequences), and methods of producing and using the same.
2. Discussion of the Background
Nisin (a 34-residue long peptide produced by Lactococcus lactis) and subtilin (a 32-residue long peptide produced by Bacillus subtilis) are the most thoroughly-studied examples of lantibiotics. Lantibiotics are ribosomally-synthesized antimicrobial peptides characterized by the presence of unusual lanthio and dehydro amino acid residues. The structures of nisin and subtilin are shown in FIG. 1. Their biosynthesis involves several post-translational modifications; e.g., dehydration of serines and threonines, formation of thioether crosslinkages between dehydro residues and cysteines, translocation, removal of a leader sequence, and/or release of the mature antimicrobial peptide into the extracellular medium (reviewed in refs. 1-3 below).
Gene-encoded antimicrobial peptides constitute a family of natural products whose known members are expanding rapidly in number and diversity, and are produced by many kinds of organisms, ranging from bacteria to eukaryotes, including mammals (1, 4-6). The ubiquity of anti-microbial peptides among widely diverged organisms implies that the peptides have been subject to many different strategies for achieving their antimicrobial properties, some of which are quite different from the properties and corresponding mechanisms of classical antibiotics such as penicillin. It may therefore be possible to supplement the arsenal of therapeutic antimicrobial agents that has been depleted as a result of the evolution of resistance among microbes.
An advantage unique to gene-encoded antimicrobial peptides is that their structures can be readily manipulated by mutagenesis, which provides a facile means for constructing and producing the large numbers of structural analogs needed for structure-function studies and rational design. Whereas this advantage is shared by all gene-encoded antimicrobial peptides, the lantibiotics are unique in possessing the unusual dehydro and lanthio amino acid residues, which are absent from magainins (7-9), defensins (10-13), or cecropins (14, 15). This means that the lantibiotics offer chemical and physical properties, and hence biological activities, that are not attainable by polypeptides that lack these residues.
For example, the dehydro residues (dehydroalanine, or "Dha," and dehydrobutyrine, or "Dhb") are electrophiles, whereas none of the 20 common natural amino acids is electrophilic. The thioether crosslinkages of lantibiotics are more resistant to cleavage or breakage than the more common disulfide bridge of proteins lacking lanthio residues. For example, a thioether crosslinkage can survive reducing conditions and extremes of pH and temperature better than a disulfide bridge (16).
A concern when making mutants of lantibiotics is the effect of the mutations on the post-translational modification process, because a mutation that disrupts processing makes the biosynthesis of the corresponding mature lantibiotic peptide impossible. All known lantibiotic prepeptides contain an N-terminal region that is cleaved during maturation. For the Type A lantibiotics (e.g. nisin, subtilin, epidermin), this leader region is highly conserved (17). Participation of the leader sequence in the orchestration of post-translational modification and secretion has been proposed (17, 18).
Certain mutations in the leader region of the nisin prepeptide have rendered the cell incapable of nisin production (19), whereas many mutations in the structural region of several lantibiotics do not disrupt processing (e.g., U.S. Pat. No. 5,516,682 and refs. 20 and 21)). When the complete nisin prepeptide consisting of the nisin leader region and the nisin structural region (N.sub.L -Nis.sub.1-34) was expressed in a subtilin-producing cell, no nisin-related peptide products were detectable (22, 23). However, when a chimera consisting of the subtilin leader region and the nisin structural region (S.sub.L -Nis.sub.1-34) was expressed in a subtilin-producing cell, an inactive nisin-like peptide was produced in which the leader region had been correctly cleaved and which contained a full complement of unusual amino acids (22). The lack of activity was attributed to the formation of incorrect thioether crosslinkages (22).
Similarly, when a prepeptide consisting of a subtilin leader region and a nisin structural region was expressed in a nisin-producing cell, the nisin structural region contained the unusual amino acids, but the leader was not cleaved (24). It has also been reported that expression of a prepeptide consisting of the nisin structural region fused to a subtilin-nisin chimeric leader region, S.sub.L(1-7) -N.sub.L(8-23) -Nis.sub.(1-34), forms active nisin when expressed in a subtilin-producing cell (23).
These results imply that subtilin processing strains such as B. subtilis are not capable of recognizing the nisin prepeptide (which is ordinarily expressed in Lactococcus lactis) and converting it to nisin. However, the subtilin processing machinery will perform modification reactions on the nisin structural peptide if it is attached to a subtilin leader region, although the modifications seem to be misdirected so that active nisin is not always produced. Finally, the subtilin processing machinery will produce active nisin if the leader region is an appropriate combination of subtilin leader and nisin leader sequences.
Lantibiotics are known to be useful bacteriocides and food preservatives. Methods of producing lantibiotics are also known. Lantibiotics offer the advantages of peptide products, in that they are more easily digested, tolerated and/or secreted by humans, other mammals and other animals which may ingest the same than are some so-called "small molecule" preservatives. Therefore, a need is felt for new lantibiotics having improved chemical, physical and/or biological properties and for improved methods of producing the same.